Project Summary Whereas phosphorylation of serine, threonine and tyrosine are exceedingly well characterized, relatively little is known about phosphorylation of histidine (His), which may account for as much as ~6% of all incorporation of phosphate into mammalian proteins. We have provided genetic and biochemical evidence that the histidine kinase, NDPK-B, and the histidine phosphatases (PTases), PHPT1 and (PGAM5, regulate the activity of the Ca2+-activated K+ channel KCa3.1 by reversible His phosphorylation, and thereby the activation of CD4 T and mast cells. While NDPK-B His phosphorylates and activates KCa3.1, PGAM5 and PHPT1 inhibit KCa3.1 His phosphorylation by specifically dephosphorylating and inhibiting NDPK-B and KCa3.1 respectively. Using recently developed monoclonal antibodies to 1- and 3-phospho-Histidine (pHis), we demonstrate for the first time the regulation of histidine phosphorylation in vivo in mammalian cells, which we in turn linked to TCR signaling. SA1 we will build on these studies, we will assess changes in His phosphorylation of NDPK-B and KCa3.1 in the context of TCR signaling, determine how it is regulated by various signaling molecules such as PI3KC2?, whether other pHis proteins are present in T cells and/or regulated by TCR signaling, and the specific role for PGAM5, PHPT1 and NDPK-B to modulate changes in pHis proteins. We have found that the ?24 cleaved PGAM5-L isoform is most critical to dephosphorylate NDPK-B. We will determine whether the ?24 cleaved PGAM5-L isoform negatively regulates CD4 T cells in vivo, whether the amount of this isoform changes following TCR stimulation, and the intramembranous proteases that mediates cleavage in CD4 T cells. We also identified a critical role for histidine phosphorylation in pancreatic ? cell function. We found that ? cells from PHPT1-/- mice have electrical properties similar to those of patients with mutations in KATP channel subunits and KATP channel-/- mice. The defect in PHPT1-/- ? cells can be explained by the failure of KATP channels to relocalize from an intracellular compartment to the plasma membrane (PM) in response to low glucose and leptin and we have now linked the defect in PHPT1-/- ? cells to impaired activation of transient receptor potential channel 4 (TRPC4). Our hypothesis is that reversible His phosphorylation of TRPC4 by PHPT1, NDPK-B, PGAM5 regulates TRPC4 channel activity in a similar manner to KCa3.1, albeit in opposite directions; whereas His phosphorylation of KCa3.1 activates, His phosphorylation of TRPC4 inhibits. In SA 2, we will determine if PHPT1, NDPK-B, and PGAM5 regulate His phosphorylation of TRPC4 in a manner similar to KCa3.1, their role in KATP channel trafficking and TRPC4 activation, and whether decreased KATP trafficking to the PM in TRPC4-/- and PGAM5-/- mice leads congenital hyperinsulinemia hypoglycemia that is similar to PHPT-/- mice and patients with CHI. We will then extend these studies to human ? cells and assess the potential relevance of these molecules to human disease that include CHI and type 2 diabetes mellitus.